Re-scan microscope system and method

ABSTRACT

A re-scan microscope for forming an image of a sample is disclosed. The system comprises an illumination optical system for directing, and optionally focusing, illumination light at the sample herewith providing an illumination light spot at the sample. The illumination light spot causes emission light from the sample. The microscope system further comprises a detection optical system for focusing at least part of the emission light onto an imaging plane of an imaging system herewith causing an emission light spot on the imaging plane. The microscope system also comprises a rotatable element for, when rotating, moving the illumination light spot over and/or through the sample and simultaneously moving the emission light spot over said imaging plane of the imaging system. The rotatable element comprises at least two reflective surfaces.

CROSS-REFERENCE TO RELATED APPLICATION

This Application is a Section 371 National Stage Application ofInternational Application No. PCT/NL2020/050420, filed Jun. 26, 2020 andpublished as WO/2020/263094 A1 on Dec. 30, 2020, in English, thecontents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This disclosure relates to a re-scan microscope system. In particular toa re-scan microscope system that comprises a rotatable element that isconfigured to rotate over 360 degrees.

BACKGROUND

A re-scan confocal microscope is known from De Luca G M, Breedijk R M,Brandt R A, et al. Re-scan confocal microscopy: scanning twice forbetter resolution. Biomed Opt Express. 2013; 4(11):2644-2656. Published2013 Oct. 25. doi:10.1364/BOE.4.002644, hereinafter referred to as “DeLuca”. This microscope has two units: 1) a standard confocal microscopewith a set of scanning mirrors which have double function: scanning theexcitation light and de-scanning the emission light, and 2) are-scanning unit that “writes” the light that passes a pinhole onto aCCD-camera. By controlling the ratio of the angular amplitude of therespective scanning mirror and rescanning mirror, the properties of themicroscope can be controlled.

In such a re-scan system it is important that the scanning andrescanning mirrors perform synchronized movements. Preferably, eachsweep of the scanning mirror exactly begins, respectively ends, at thesame time as a corresponding sweep of the rescanning mirror begins,respectively ends. If the scanning and re-scanning mirror moveasynchronously, the obtained image will have a low quality. As will beunderstood, at higher scan speeds, the mirrors move faster and theacceptable margin of error for the synchronization becomes smaller.Since the degree of synchronization of the mirrors in the system of DeLuca is limited, the scan speed is also limited.

Therefore, there is a desire in the art for a re-scan microscope thatcan scan a sample at higher scan speeds while maintainingsynchronization of the scanning and re-scanning mirrors.

Gregor et al, Image scanning microscopy, Current Opinion in ChemicalBiology 2019, Volume 51, page 74-83 discloses the basic principles ofImage Scanning Microscopy and in particular discloses a re-scan imagescanning microscope that comprises a scanning mirror and re-scanningmirror.

Li et al, The Journal of Investigative Dermatology, volume 125:798-804,2005 discloses a dual contrast confocal microscope. Herein, a reflectedlight channel is formed by a laser diode aimed through a beamsplittercube and onto a rotating polygonal mirror for fast scanning. The lightthen goes to a galvo scanner and the objective lens. The incident lightis scattered by the tissue and the reflected light retraces the opticalpath. A bs diverts the reflected light to a confocal detector. Thefluorescence channel has an argon laser (Ar+) coupled to the scanner; adichroic mirror (dm) diverts the excitation light to a combiningdichroic mirror (cd) that aligns the fluorescence excitation light (488nm) to the reflectance light (830 nm). After the cd the two beams sharethe same optical path to the sample and back. The returning fluorescencesignal is diverted by the combining dichroic (cd) through the dichroicmirror (dm), onto the barrier filter (bf) that eliminates any remainingexcitation light, and to a second confocal detector (FCM).

Hari P. Paudel. Yookyung Jung, Anthony Raphael. Clemens Alt, Juwell Wu,Judith Runnels, and Charles P. Lin “In vivo flow cytometry for bloodcell analysis using differential epi-detection of forward scatteredlight”, Proc. SPIE 10497. Imaging. Manipulation. and Analysis ofBiomolecules, Cells, and Tissues XVI, 104970G (20 Feb. 2018) discloses aflow cytometer comprising a 36 facet polygon scanner.

SUMMARY

To this end a re-scan microscope for forming an image of a sample isdisclosed. The system comprises an illumination optical system fordirecting, and optionally focusing, illumination light at the sampleherewith providing an illumination light spot at the sample. Theillumination light spot causes emission light from the sample. Themicroscope system further comprises a detection optical system forfocusing at least part of the emission light onto an imaging plane of animaging system herewith causing an emission light spot on the imagingplane. The microscope system also comprises a rotatable element for,when rotating, moving the illumination light spot over and/or throughthe sample and simultaneously moving the emission light spot over saidimaging plane of the imaging system. The rotatable element comprises atleast two reflective surfaces.

Because a single rotatable element is used for both scanning theillumination light spot over and/or through the sample and re-scanningthe emission light spot over the imaging plane, good synchronizationbetween the respective movements of the illumination light spot andemission light spot can be readily achieved, irrespective of the angularvelocity with which the rotatable element is rotating for moving thelight spots. Therefore, the re-scan microscope system enables to scan athigh speeds while still maintaining synchronization.

The at least two reflective surfaces are, optionally, non-parallel,which may be understood as that they are oriented at a nonzero andnon-180 degrees angle with respect to each other. A normal vector of areflective surface may be understood to be a vector that isperpendicular to the reflective surface, has its initial point at thereflective surface and points outwards, i.e. to the side of the surfacewhere the incident and reflected light are. The angle between two normalvectors of two respective reflective surfaces may be understood to bethe smallest angle that the two vectors would make when they would beboth positioned in the standard position. i.e. with their initial pointsat the origin in the Cartesian coordinate system. In general, an anglebetween two surfaces may be understood to be the angle between theirrespective normal vectors. Thus, stating that two surfaces are orientedat an angle of a particular size with respect to each other may beunderstood to be equivalent to stating that the angle between therespective normal vectors of the two surfaces is of said particularsize.

A reflective surface may be understood to relate to any reflectiveelement, e.g. mirrors, splitters, prisms, etc. A reflective multilayercoating is for example also to be regarded as a reflective surface asused herein. A reflective surface may be understood to provide anintended reflection. A reflective surface as used herein may beconfigured to partially reflect light. In an example, a reflectivesurface only reflects a particular wavelength range and/or only aparticular polarization direction.

In an embodiment, the rotatable element is configured to rotate over anangle of at least 90 degrees about an axis of rotation, preferably torotate over an angle of at least 180 degrees, more preferably over anangle of at least 360 degrees, most preferably configured to rotateinfinitely, about an axis of rotation. This embodiment allows highspeed, energy efficient scanning because the rotatable element can bebrought into its initial position without having to change angularmomentum. Conventional scanning mirrors, such as the scanning mirror andre-scanning mirror of De Luca, need, after a scanning movement from aninitial scanning position to an end scanning position, to come to a haltand return to the initial scanning position. Because of this, the speedwith which these mirrors can pivot is limited. In such embodiment, thetwo reflective surfaces may or may not be parallel.

In an embodiment, the rotatable element is configured to consecutivelymake a plurality of full rotations, preferably the rotatable elementhaving a substantially constant angular momentum when making saidplurality of full rotations.

In an embodiment, the rotatable element comprises a first reflectivesurface and a second reflective surface. In such embodiment, the re-scanmicroscope system is configured such that, in use, during a rotation ofthe rotatable element, the first reflective surface reflects theillumination light throughout a first time period and changesorientation during said first time period for moving the illuminationlight spot over and/or through the sample.

Since the first and second reflective surface are part of the samerotatable element, their orientation with respect to each other andposition with respect to each other may be fixed and move together onrotation of the rotatable element. Thus, any movement of the firstreflective surface causes the first and second reflective surfaces tomove together. This holds at any angular velocity with which therotatable element is rotating. Therefore, the present re-scan microscopeenables high speed, synchronous movements of the first and secondreflective surfaces and enables to scan samples at high speeds.

It should be understood that the reflective surfaces of the rotatableelement may change orientation due to the rotation of the rotatableelement. The reflective surfaces are preferably not circumferentiallycurved with respect to the rotational axis of the rotatable element, inparticular being plane in a tangential direction with respect to therotational axis.

A reflective surface of the rotatable element reflecting theillumination light throughout a time period and changing orientationduring this time period for moving the illumination light spot overand/or through the sample may be referred to as the reflective surfaceperforming a scanning function. Further, in this situation, theillumination light may be said to be scanned by the reflective surface.

A reflective surface reflecting emission light during a time period andchanging orientation during this time period for moving the emissionlight spot over the imaging plane may be referred to as the reflectivesurface performing a re-scanning function. Further, in this situation,the emission light may be said to be re-scanned by the reflectivesurface.

Preferably, the rotational axis of the rotatable element does not crossthe first nor the second reflective surface.

In an embodiment, the re-scan microscope system is configured such that,in use, during said first time period, the second reflective surfacereflects the emission light and changes orientation during said firsttime period for moving the emission light spot over the imaging plane.In such embodiment, it may be understood as that the first reflectivesurface is used as a scanning mirror (and possibly de-scanning mirror)while the second reflective surface is used as a re-scanning mirror.

Such embodiment enables good separation of illumination light andemission light. It should be understood that some of the illuminationlight that is incident onto the sample may be scattered back. Thisbackscattered illumination light may be significantly stronger, evenseveral orders of magnitude stronger, e.g. a million times stronger,than the emission light. Hence, it is desired that such backscatteredillumination light does not reach the imaging system. This can bereadily achieved by using one mirror as scanning mirror and another asre-scanning mirror.

In such embodiment, the first and second reflective surface may beoriented at a nonzero and non-180 degrees angle with respect to eachother. This allows illumination light and emission light to approach therotatable element while traveling substantially in parallel directions.Due to the non-zero angle of the reflective surfaces, the illuminationlight and emission light can for example be reflected in different,optionally even substantially opposite directions, the illuminationlight towards the sample and the emission light towards the imagingsystem. This allows to position the imaging system and sample atdifferent, e.g. opposite, sides of the rotatable element without havingto introduce further mirrors for guiding the illumination light towardsthe sample and/or for guiding the emission light towards the imagingsystem, thus allowing compact set-ups. Further, because the anglebetween the first and second reflective surfaces is non-180 degrees,there is no need to completely reroute the emission light, after it hasbeen de-scanned by the rotatable element, around the rotatable element,which would require additional mirrors and relatively long optical pathsbetween such mirrors.

In an embodiment, the re-scan microscope system is configured such that,in use, at least part of the illumination light, after it has beenscanned by a reflective surface of the rotatable element, is notincident on a mirror before being incident on the sample. Thisembodiment also allows for compact optical setups.

In an embodiment, the re-scan microscope system is configured such that,in use, the emission light, after it has been re-scanned by a reflectivesurface of the rotatable element, is not incident on a mirror beforebeing incident on the imaging plane of the imaging system. Thisembodiment also allows for compact optical setups.

In an embodiment wherein the first reflective surface is used as ascanning mirror while the second reflective surface is used as are-scanning mirror, the re-scan microscope system is configured suchthat, in use, during the rotation of the rotatable element, the secondreflective surface reflects the illumination light throughout a secondtime period and changes orientation during said second time period formoving the illumination light spot over and/or through the sample andconfigured such that, in use, during said second time period, a furtherreflective surface of the rotatable element reflects the emission lightand changes orientation during said second time period for moving theemission light spot over the imaging plane. Herein, the furtherreflective surface may be the first reflective surface.

In such embodiment, the second reflective surface, during a rotation ofthe rotatable element, both performs a scanning function and arescanning function and is thus efficiently used.

The first and second time periods may be understood to benon-overlapping time periods. The first and second reflective surfacesmay be understood to be different surfaces.

In an embodiment, the rotatable element may comprise a third reflectivesurface and the re-scan microscope may be configured such that, in use,during the rotation of the rotatable element, the third reflectivesurface reflects the illumination light throughout a third time periodand changes orientation during said third time period for moving theillumination light spot over and/or through the sample and configuredsuch that, during said third time period, another reflective surface ofthe rotatable element reflects the emission light and changesorientation during said third time period for moving the emission lightspot over the imaging plane. In such embodiment, the first and thirdreflective surface are non-parallel and, optionally, the first andsecond reflective surface are oriented at an angle of 180 degrees withrespect to each other. The third time period may occur between the firstand second time period mentioned above.

In such embodiment, the angle over which the rotatable element has torotate before a next line is scanned over the sample/imaging plane isrelatively small, which benefits the scan speed.

In an embodiment, the re-scan microscope system is configured such that,in use, during said first time period, the first reflective surfacereflects the emission light and changes orientation during said firsttime period for moving the emission light spot over the imaging planeand such that, in use, during the rotation of the rotatable element, thesecond reflective surface reflects the illumination light throughout asecond time period and changes orientation during said second timeperiod for moving the illumination light spot over and/or through thesample.

In such embodiment, it may be understood as that the first reflectivesurface is simultaneously used both as a scanning mirror (andde-scanning mirror) and re-scanning mirror.

In such embodiment, the first and second reflective surface may beoriented at a nonzero and non-180 degrees angle with respect to eachother, so that the rotatable element does not have to rotate over 180degrees after the first reflective surface has performed the doublescanning and re-scanning function before the second reflective surfacecan perform the double scanning and re-scanning function, which enableshigh scan speeds. In particular, more scan lines can be written per fullrotation of the rotatable element.

In an embodiment, wherein the first reflective surface is simultaneouslyused both as a scanning mirror and re-scanning mirror, the re-scanmicroscope system is configured such that, in use, during said secondtime period, the second reflective surface reflects emission light andchanges orientation during said second time period for moving theemission light spot over the image plane.

In such embodiment, both the first and second reflective surface eachsimultaneously perform a double scanning and re-scanning function duringa rotation the rotatable element, the first reflective surface duringthe first time period and the second reflective surface during thesecond time period.

In an embodiment, the rotatable element comprises a rotatable polygonscanner comprising a plurality of reflective facets, wherein the firstreflective surface is a first facet of the plurality of reflectivefacets and the second reflective surface is a second facet of theplurality of reflective facets.

The polygon scanner may have any number of facets, each facet being areflective surface that, during a rotation, performs a scanning functionand/or re-scanning function.

In an embodiment, the re-scan microscope system is configured to movethe illumination light spot over and/or through the sample at a firstvelocity and move the emission light spot over the imaging plane at asecond velocity, such that the second velocity is different from,preferably higher than, a baseline velocity. The baseline velocity isdefined as the first velocity multiplied by the optical magnification ofthe re-scan microscope system. This embodiment allows to improve theresolution of the formed image of the sample.

The optical magnification of the microscope system may be understood tobe the magnification of the sample that arises because of the differentlenses in the microscope system and does not include any magnificationthat arises because of the second velocity being higher than thebaseline velocity. The latter magnification may be referred to asmechanical magnification as opposed to optical magnification. As such,the optical magnification of the microscope system that defines thebaseline velocity may be understood to relate to the magnification ofthe illumination light spot, for example in the sense that it relates toa ratio between a dimension, e.g. a diameter, of the emission light spotas projected onto the imaging plane of the imaging system and acorresponding dimension, e.g. also the diameter, of the illuminationlight spot at the sample.

In an embodiment, the second velocity is approximately twice as high asthe baseline velocity. This advantageously optimizes the resolution ofthe to be formed image.

In an embodiment, the re-scan microscope system comprises an objectiveconfigured to gather the emission light from the sample and focus theemission light on a primary image plane of the re-scan microscopesystem. In such embodiment, the detection optical system is configuredto image images in the primary image plane onto the imaging plane of theimaging system. The primary image plane may be understood to be thefirst image plane that the emission light passes on its way to theimaging plane of the imaging system.

In one embodiment, the detection optical system is configured to imageimages in the primary image plane onto the imaging plane of the imagingsystem with an optical magnification of approximately 0.5. Thisembodiment allows to obtain a sweep factor of 2 as described in De Lucawhile the scanning and re-scanning mirrors have equal scanningamplitudes.

In an embodiment, the re-scan microscope system is configured such that,in use, an angle between a travel direction of the illumination lightthat is incident on the rotatable element and a travel direction of theemission light that is incident on the rotatable element for moving theemission light spot over the imaging plane is less than 90 degrees,preferably less than 60 degrees, more preferably less than 30 degrees,most preferably approximately zero degrees. This embodiment allows avery compact setup of the re-scan microscope system.

A travel direction of light may be represented by a vector. The anglebetween two travel directions may be understood to refer to the smallestangle that the respective vectors of the travel directions would makewhen the vectors would be positioned in the standard position. i.e. withtheir initial points at the origin in the Cartesian coordinate system.

In an embodiment, the rotatable element is configured to reflect theillumination light in a first variable direction and to reflect theemission light for moving the emission light spot over the imaging planein a second variable direction, wherein the angle between the first andsecond variable direction is larger than 90 degrees, preferably largerthan 120 degrees, more preferably larger than 150 degrees, mostpreferably approximately 180 degrees.

This embodiment advantageously enables to position the imaging systemand sample at opposite sides of the rotatable element, which allows fora compact optical setup.

Preferably, the first and second variable directions lie in a radialsurface around the rotational axis of the rotatable element or makerelatively small angles, such as at most 10 degrees, with respect tosuch radial surface. A radial surface may be understood to be a surfacethat is perpendicular to the axis of rotation. In other words, the firstdirection, respectively second direction, preferably does not vary suchthat the angle between a radial surface around the rotational axis ofthe rotatable element and the first, respectively second, directionbecomes higher than 10 degrees. Again, a direction can be represented bya vector. The angle between a direction and a surface may be understoodto be the smallest angle that the vector representing this directionmakes with the surface when the initial point of the vector would bepositioned at the surface.

In an embodiment, the microscope system comprises an aperture, such as apinhole or slit, for passing emission light and an optical system forfocusing the emission light onto the aperture. This embodiment enablesthe re-scan microscope system to perform optional sectioning.

One aspect of this disclosure relates to a method for forming an imageof a sample using a re-scan microscope system. The re-scan microscopesystem comprises an illumination optical system for directing, andoptionally focusing, illumination light at the sample therewithproviding an illumination light spot at the sample, the illuminationlight spot causing emission light from the sample. The re-scanmicroscope system further comprises a detection optical system focusingat least part of the emission light onto an imaging plane of an imagingsystem herewith causing an emission light spot on the imaging plane. There-scan microscope system further comprises a rotatable elementcomprising at least two non-parallel reflective surfaces. The methodcomprises rotating the rotatable element for moving the illuminationlight spot over and/or through the sample and simultaneously moving theemission light spot over said imaging plane of the imaging system. There-scan microscope system may be any of the re-scan microscope systemsas described herein.

Optionally, the method comprises rotating the rotatable element over anangle of at least 90 degrees about an axis of rotation, preferably overan angle of at least 180 degrees, more preferably over an angle of atleast 360 degrees, most preferably over multiple full rotations.

Optionally, the method comprises generating the illumination light.

Optionally, the method comprises incrementally rotating a further scansystem, such as a y-axis scan system, multiple times during one fullrotation of the rotatable element so that a desired area of the sampleis scanned, for example as depicted in FIGS. 6A and 6B.

One aspect of this disclosure relates to a computer-implemented methodcomprising the step of causing the re-scan microscope system to performthe method for forming an image of a sample as described herein.

One aspect of this disclosure relates to a computer comprising acomputer readable storage medium having computer readable program codeembodied therewith, and a processor, preferably a microprocessor,coupled to the computer readable storage medium, wherein responsive toexecuting the computer readable program code, the processor isconfigured to perform the method computer-implemented method asdescribed herein.

One aspect of this disclosure relates to a computer program or suite ofcomputer programs comprising at least one software code portion or acomputer program product storing at least one software code portion, thesoftware code portion, when run on a computer system, being configuredfor executing the computer-implemented method described herein.

One aspect of this disclosure relates to a non-transitorycomputer-readable storage medium storing at least one software codeportion, the software code portion, when executed or processed by acomputer, is configured to perform the computer-implemented describedherein.

As will be appreciated by one skilled in the art, aspects of the presentinvention may be embodied as a system, a method or a computer programproduct. Accordingly, aspects of the present invention may take the formof an entirely hardware embodiment, an entirely software embodiment(including firmware, resident software, micro-code, etc.) or anembodiment combining software and hardware aspects that may allgenerally be referred to herein as a “circuit,” “module” or “system.”Functions described in this disclosure may be implemented as analgorithm executed by a processor/microprocessor of a computer.Furthermore, aspects of the present invention may take the form of acomputer program product embodied in one or more computer readablemedium(s) having computer readable program code embodied. e.g., stored,thereon.

Any combination of one or more computer readable medium(s) may beutilized. The computer readable medium may be a computer readable signalmedium or a computer readable storage medium. A computer readablestorage medium may be, for example, but not limited to, an electronic,magnetic, optical, electromagnetic, infrared, or semiconductor system,apparatus, or device, or any suitable combination of the foregoing. Morespecific examples of a computer readable storage medium may include, butare not limited to, the following: an electrical connection having oneor more wires, a portable computer diskette, a hard disk, a randomaccess memory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), an optical fiber, a portablecompact disc read-only memory (CD-ROM), an optical storage device, amagnetic storage device, or any suitable combination of the foregoing.In the context of the present invention, a computer readable storagemedium may be any tangible medium that can contain, or store, a programfor use by or in connection with an instruction execution system,apparatus, or device.

A computer readable signal medium may include a propagated data signalwith computer readable program code embodied therein, for example, inbaseband or as part of a carrier wave. Such a propagated signal may takeany of a variety of forms, including, but not limited to,electro-magnetic, optical, or any suitable combination thereof. Acomputer readable signal medium may be any computer readable medium thatis not a computer readable storage medium and that can communicate,propagate, or transport a program for use by or in connection with aninstruction execution system, apparatus, or device.

Program code embodied on a computer readable medium may be transmittedusing any appropriate medium, including but not limited to wireless,wireline, optical fiber, cable, RF, etc., or any suitable combination ofthe foregoing. Computer program code for carrying out operations foraspects of the present invention may be written in any combination ofone or more programming languages, including an object orientedprogramming language such as Java™, Smalltalk, C++ or the like andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The program codemay execute entirely on the user's computer, partly on the user'scomputer, as a stand-alone software package, partly on the user'scomputer and partly on a remote computer, or entirely on the remotecomputer or server. In the latter scenario, the remote computer may beconnected to the user's computer through any type of network, includinga local area network (LAN) or a wide area network (WAN), or theconnection may be made to an external computer (for example, through theInternet using an Internet Service Provider).

Aspects of the present invention are described below with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of thepresent invention. It will be understood that each block of theflowchart illustrations and/or block diagrams, and combinations ofblocks in the flowchart illustrations and/or block diagrams, can beimplemented by computer program instructions. These computer programinstructions may be provided to a processor, in particular amicroprocessor or a central processing unit (CPU), of a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer, other programmable dataprocessing apparatus, or other devices create means for implementing thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

These computer program instructions may also be stored in a computerreadable medium that can direct a computer, other programmable dataprocessing apparatus, or other devices to function in a particularmanner, such that the instructions stored in the computer readablemedium produce an article of manufacture including instructions whichimplement the function/act specified in the flowchart and/or blockdiagram block or blocks.

The computer program instructions may also be loaded onto a computer,other programmable data processing apparatus, or other devices to causea series of operational steps to be performed on the computer, otherprogrammable apparatus or other devices to produce a computerimplemented process such that the instructions which execute on thecomputer or other programmable apparatus provide processes forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks.

The flowchart and block diagrams in the figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof code, which comprises one or more executable instructions forimplementing the specified logical function(s). It should also be notedthat, in some alternative implementations, the functions noted in theblocks may occur out of the order noted in the figures. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams and/or flowchart illustrations,and combinations of blocks in the block diagrams and/or flowchartillustrations, can be implemented by special purpose hardware-basedsystems that perform the specified functions or acts, or combinations ofspecial purpose hardware and computer instructions.

Moreover, a computer program for carrying out the methods describedherein, as well as a non-transitory computer readable storage-mediumstoring the computer program are provided. A computer program may, forexample, be downloaded (updated) to the existing data processing systemsor be stored upon manufacturing of these systems.

Elements and aspects discussed for or in relation with a particularembodiment may be suitably combined with elements and aspects of otherembodiments, unless explicitly stated otherwise. Embodiments of thepresent invention will be further illustrated with reference to theattached drawings, which schematically will show embodiments accordingto the invention. It will be understood that the present invention isnot in any way restricted to these specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention will be explained in greater detail byreference to exemplary embodiments shown in the drawings, in which:

FIG. 1 schematically shows a re-scan microscope system according to anembodiment;

FIG. 2 schematically shows, in three dimensions, a re-scan microscopesystem according to an embodiment;

FIGS. 3A-3C illustrate scanning and re-scanning as performed by therotatable element during a rotation according to an embodiment, whereinthe scanning and re-scanning functions are performed by differentreflective surfaces;

FIGS. 4A-4C illustrate scanning and re-scanning as performed by therotatable element during a rotation according to an embodiment, whereinthe scanning and re-scanning functions are performed by a singlereflective surface;

FIGS. 5A-5C illustrate scanning and re-scanning as performed by therotatable element during a rotation according to an embodiment, whereinthe scanning and re-scanning functions are performed by two parallelreflective surfaces;

FIGS. 6A-6B illustrate the movement of illumination light in a primaryimage plane and corresponding movement of the emission light spot in theimaging plane of the imaging system according to an embodiment;

FIGS. 7A-7B show rotatable elements according to two differentembodiments;

FIG. 8 shows a data processing system according to an embodiment thatmay be used in a re-scan microscope system according to an embodiment.

DETAILED DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numerals indicate identical orsimilar elements.

FIG. 1 schematically shows a re-scan microscope system 10 for forming animage of a sample 32 according to one embodiment. A light source, suchas a laser 11, generates illumination light 16. An optical fiber cable12 may be used to guide the illumination light 16 from light source 11to the re-scan microscope system 10. A collimating lens 14 converts thedivergent illumination light as output by the optical fiber cable 12into a parallel beam.

The system 10 comprises an illumination optical system for directing theillumination light 16 at the sample. In the depicted embodiment, theillumination optical system comprises a dichroic mirror 18, a y-axisscan system 20 a, relay optics comprising lenses 22 a and 22 b, a firstreflective surface 24 a, such as a mirror, of a rotatable element 25, ascan lens 26, a tube lens 28 and an objective 30. The illuminationoptical system is configured to direct, and optionally focus, theillumination light 16 at the sample. Herewith, an illumination lightspot 34 is provided at the sample 32. In this example, the illuminationlight spot is positioned in plane 36 through the sample 32. The samplemay be fluorescently labelled and may be a biological sample.

The illumination light spot 34 at the sample 32 causes emission light 46from the sample. In one example, the illumination light spot causesoptical excitations in the sample 32, which optical excitations, upondecaying to lower energy levels, cause the emission light. In thisexample, the illumination light 16 may be regarded as excitation lightand the emission light as fluorescent light. In another example, theillumination light spot causes emission light from the sample 32 in thesense that the illumination light is reflected by the sample 34. Thereflected light may also be regarded as emission light 46, in particularas reflection emission light.

The re-scan microscope system 10 further comprises a detection opticalsystem for focusing the emission light onto an imaging plane 56 of animaging system 52. Herewith an emission light spot 54 is caused on theimaging plane 56. The imaging system may be a camera, such as a CCDcamera. The imaging plane preferably comprises a plurality of pixelsthat are arranged in a predefined manner. e.g. in a 2D lattice. In thedepicted embodiment, the detection optical system comprises scan lens26, reflective surface 24 a, relay lenses 22 a and 22 b, y-axis scansystem 20 a, mirror 38, a pinhole 42, lenses 40 a and 40 b positioned oneither side of pinhole 42, mirror 44, y-axis scan unit 20 b, relaylenses 22 c and 22 d, a second reflective surface 24 g and re-scan lens50. However, it should be appreciated that the depicted detectionoptical system is merely an example and that the detection opticalsystem used for the invention may in principle comprise less, or more,optical elements, such as mirrors and lenses, than depicted in FIG. 1 .

The depicted embodiment comprises a dichroic mirror 18 that isconfigured to reflect illumination light 16 travelling towards thesample 32 and to pass emission light 46 travelling from sample 32 to theimaging system 52.

The rotatable element 25 is configured to rotate over an angle of atleast 360 degrees and herewith move the illumination light spot 34,provided by the illumination optical system, over and/or through thesample 32. In the depicted embodiment, the illumination light spot 34moves over plane 36 that lies through sample 32. The rotatable element25 also performs another function. Due to its rotation, the rotatableelement 25 namely simultaneously moves the emission light spot 54,caused by the detection optical system, over the imaging plane 56. Inone embodiment, the rotatable element 25 is configured to consecutivelymake a plurality of full rotations. Preferably, the rotatable elementcan rotate infinitely.

For each rotation of the rotatable element, each reflective surface ofthe rotatable element may perform a scanning function and/or are-scanning function at least once, preferably once.

It should be understood that the reflective surface 24 a may both scanthe illumination light so that the illumination light spot moves overand/or through the sample 32 as well as de-scan the emission light 46from the sample 32 so that a static light beam of emission light isformed between reflective surface 24 a and reflective surface 24 g.Static may be understood as not moving with respect to the rotationalaxis 60 of the rotatable element 25.

Each reflective surface 24 a-24 h of the rotatable element 25 has anormal vector. Preferably, the respective normal vectors of thereflective surfaces lie in the same plane. Preferably, this plane isperpendicular to the axis of rotation of the rotatable element. In oneembodiment, the normal vectors of the reflective surfaces make equalangles with respect to each other.

In an embodiment, adjacent reflective surfaces of the rotatable elementare oriented at an angle with respect to each other that is smaller than90 degrees, i.e. the respective normal vectors of the adjacent surfacesmake an angle with respect to each other that is smaller than 90degrees. Note that in the depicted example, the normal vectors aredirected away from the rotational axis of the rotatable element.

The reflective surfaces may be fixed onto the rotatable element. In anembodiment, the orientation of each of the reflective surfaces withrespect to the rotatable element may be adaptable in order to easealignment of the optical system.

In an embodiment, the rotatable element 25 comprises a rotatable polygonscanner comprising a plurality of reflective facets 24 a. 24 g, whereinthe first reflective surface 24 a is a first facet 24 a of the pluralityof reflective facets and the second reflective surface 24 g is a secondfacet 24 g of the plurality of reflective facets. In principle, thepolygon scanner may comprise any number of facets. In the depictedembodiment, other facets are indicated by 24 b. 24 c. 24 d, 24 e, 24 f.24 h.

The depicted re-scan microscope system 10 is configured such that, inuse, during a rotation of the rotatable element 25, the first reflectivesurface 24 a reflects the illumination light 16 throughout a first timeperiod and changes orientation during said first time period for movingthe illumination light spot 34 over and/or through the sample 32. In oneembodiment, the re-scan microscope system 10 is configured such that, inuse, during said first time period, the second reflective surface 24 greflects the emission light 46 and changes orientation during said firsttime period for moving the emission light spot 54 over the imaging plane56.

In the depicted embodiment, the re-scan microscope system 10 isconfigured such that, in use, during the rotation of the rotatableelement 25, the second reflective surface 24 g reflects the illuminationlight 16 throughout a second time period and changes orientation duringsaid second time period for moving the illumination light spot 34 overand/or through the sample 32 and configured such that, during saidsecond time period, a further reflective surface 24 e of the rotatableelement 25 reflects the emission light 46 and changes orientation duringsaid second time period for moving the emission light spot 54 over theimaging plane 56.

Preferably, the point where the illumination light 16 reflects from thefirst reflective surface lies in a focal plane of scan lens 26 so thatthe illumination light spot 34 neatly moves over plane 36. Also,preferably, the point where the emission light reflects from the secondreflective surface lies in a focal plane of re-scan lens 50 so that thefocused emission light spot 54 neatly moves over imaging plane 56. Thesame holds, respectively, for the reflective points of the illuminationand emission light on the y-axis scanning mirrors 20 a and 20 b. Ifrequired, these points may be “optically” positioned in the focal planeof the scan lens 26 and re-scan lens 50, respectively, using relayoptics known in the art. In the depicted embodiment, relay lenses 22 aand 22 b are configured to optically position the illumination light'sreflective point on the y-axis scanner 20 a in the focal plane of scanlens 26 and relay lenses 22 c and 22 d to optically position theillumination light's reflective point on the y-axis scanner 20 b in thefocal plane of re-scan lens 50. To summarize, both the reflective pointon the first reflective surface 24 a and the reflective point on they-axis scanning system 20 a are preferably in a conjugate plane of theback focal plane of the scan lens 26. Also, both the reflective point onthe second reflective surface 24 g and the reflective point on they-axis scanning system are preferably in a conjugate plane of the backfocal plane of the re-scan lens 50.

In the depicted embodiment, the re-scan microscope system 10 isconfigured to move the illumination light spot 34 over and/or throughthe sample 32 at a first velocity, v₁, and move the emission light spot54 over the imaging plane at a second velocity, v₂, such that the secondvelocity is different from, preferably higher than, a baseline velocity.The baseline velocity is defined as the first velocity multiplied by theoptical magnification of the re-scan microscope system.

In the depicted embodiment, the microscope system 10 comprises anobjective 30 configured to gather the emission light 46 from the sample32 and focus the emission light 46 on a primary image plane 27 of there-scan microscope system 10. In the depicted embodiment, the detectionoptical system is configured to image images in the primary image plane27 onto the imaging plane 56 of the imaging system 52.

The optical magnification of the re-scan microscope system 10 isindependent of the respective velocities with which the illuminationlight spot 34 and the emission light spot 54 move over the sample resp,the imaging plane 56. The optical magnification of the re-scanmicroscope system may be understood to be determined by the opticalmagnification provided by the combination of objective 30 and tube lens28, referred to as M_(micr) in De Luca, and the optical magnification M₂of the detection optical system. The optical magnification of thedetection optical system is determined by scan lens 26, relay lenses 22a-22 c, lenses 40 a and 40 b and re-scan lens 50. Assuming that therelay lenses 22 a-22 d have equal focal lengths, the opticalmagnification M₂ of the detection optical system is given by

M ₂=(f _(40a) ×f ₅₀)/(f ₂₆ ×f _(40b)), wherein f _(40a) denotes thefocal length of lens 40 a, etc.

It should be appreciated that the velocity of the illumination lightspot's image in primary image plane 27, v_(1P), is given by themultiplication of the first velocity, i.e. the actual velocity of theillumination light spot 34 over and/or through the sample, with theoptical magnification of the combination of objective 30 and tube lens28, i.e. v_(1P)=v₁×M_(micr).

The baseline velocity, v_(B), in the depicted embodiment, assuming equalfocal lengths of the relay lenses 22 a-22 d, is then given byv_(B)=v₁×M_(micr)×M₂.

In one embodiment, the second velocity is approximately twice as high asthe baseline velocity, v₂≈2×v_(B). This can for example be achieved byconfiguring the detection optical system such that it images an image inthe primary image plane 27 onto the imaging plane 56 of the imagingsystem 52 with an optical magnification of approximately 0.5, M₂≈2 0.5.In this manner, the second velocity is twice as high as the baselinevelocity if v_(1P) and v₂ are equal.

In the depicted embodiment, the system 10 comprises an aperture 42, suchas a pinhole or slit, for passing emission light 46 and an opticalsystem, in this example comprising lens 40 a, for focusing the emissionlight 46 onto the aperture 42.

Further, the depicted embodiment comprises a data processing system 100that comprises means for controlling the rotatable element and/or forcontrolling the y-axis scanning system 20 a and/or 20 b and/or forcontrolling the light source 11 and/or for controlling the imagingsystem 52.

FIG. 2 shows an embodiment of the re-scan microscope system 10 in threedimensions. For clarity, the sample and optional further lenses afterprimary image plane 27 are not shown. The illumination light 16 isincident on dichroic mirror 18, which reflects the illumination light 16towards y-axis scanning system 20 a, in the depicted embodiment towardsmirror 20 a that is configured to rotate around axis 62. Theillumination light 16 is subsequently incident on the rotatable element25, in the depicted embodiment on polygon scanner 25, that is configuredto infinitely rotate around rotational axis 60. Scan lens 26subsequently focuses the illumination light onto the primary image plane27. The further lens system that further focuses the illumination lighton the sample is not shown. However, it should be appreciated that anymovement of the illumination light over primary image plane 27corresponds to a similar movement of the illumination light spot 34 overand/or through the sample 32. To illustrate, if the illumination lightin plane 27 moves in the x-direction, then the illumination light spot34 will move over and/or through the sample in the x-direction as well,yet with a lower velocity determined by the magnification of saidfurther lens system.

The emission light 46 from the sample travels back from the sample tothe rotatable element 25, which de-scans the emission light 46 so that astatic emission light beam is formed that travels back to mirror 20 a.Mirror 20 a then reflects the emission light 46 to the dichroic mirror18 that passes the emission light 46 so that the emission light isincident on static mirror 38 and static mirror 44 that are arranged toguide the emission light 46 to scanning mirror 20 b. Scanning mirror 20b is also configured to rotate around axis 62. Preferably, the scanningmirrors 20 a and 20 b are configured to move synchronously. In oneembodiment, a single y-axis scanning mirror may be used instead of thetwo scanning mirrors 20 a and 20 b.

After being reflected by mirror 20 b, the emission light 46 is incidenton a reflective surface of the rotatable element so that the emissionlight is re-scanned. Herewith, the emission light spot 54 is moved overimaging plane 56 of imaging system 52.

A rotation of the y-axis scanning mirror 20 a causes the illuminationlight in plane 27 to move the in the y-direction as indicated and thuscauses the illumination light spot 34 to move in the y-direction as wellover and/or through the sample 32.

A rotation of the y-axis scanning mirror 20 b causes the emission lightspot 54 to move in the y-direction as indicated over the imaging plane56.

The rotatable element 25 may thus be configured to move the illuminationlight spot 34 in a particular direction. The first velocity of theillumination light spot 34 as used herein may be understood to be thevelocity component in this particular direction. Similarly, therotatable element 25 may be configured to move the emission light spot54 in a particular direction. The second velocity of the emission lightspot 54 may be understood to be the velocity component in thisparticular direction.

In the depicted embodiment, the angle between the travel direction ofthe illumination light that is incident on the rotatable element and thetravel direction of the emission light that is incident on the rotatableelement for moving the emission light spot over the imaging plane is thesame, i.e. the emission light 46 travels in the −x direction towards therotatable element and the illumination light 16 also travels in the −xdirection towards the rotatable element. Preferably, such angle is lessthan 90 degrees, preferably less than 60 degrees, more preferably lessthan 30 degrees.

In the depicted embodiment, the rotational axis is aligned with they-axis as indicated on the bottom right. A radial surface may beunderstood to be a surface that is perpendicular to the rotational axis60. In the depicted embodiment, any radial surface is thus parallel tothe x-z plane as indicated on the bottom right.

Depending on the orientation of the y-axis scanners 20 a and 20 b and ofthe reflective surfaces of the rotatable element, the respectivedirections of the re-scanned emission light 46 and of the scannedillumination light 16 varies. In particular, the angle between a radialsurface of the rotatable element and the direction of the re-scannedemission light depends on the orientation of the y-axis scanning mirror20 b, wherein the angle between a radial surface of the rotatableelement and the direction of the scanned illumination light 16 dependson the orientation of the y-axis scanning mirror 20 a. Preferably, theseangles, during the scanning of the sample are at most 10 degrees.

FIGS. 3, 4 and 5 show top views of the rotatable element at differenttime instances during a rotation. FIGS. 3A, 3B, 3C depict threerespective time instances during the first time period throughout whichthe reflective surface 24 a scans the illumination light 16.

FIG. 3 shows an embodiment wherein the re-scan microscope system 10 isconfigured such that, in use, during said first time period, the secondreflective surface 24 g reflects the emission light 46 and changesorientation during said first time period for moving the emission lightspot 54 over the imaging plane 56.

For clarity, the de-scanning of the emission light 46 is not shown.Typically, the emission light 46, before passing a dichroic mirror 18,and the illumination light 16 travel along the same path, yet inopposite directions.

In the depicted embodiment, the illumination light 16 and emission light46 travel in the same direction before being incident on the rotatableelement 25, at least as viewed from the top as depicted.

Further, in the depicted embodiment, the rotatable element is configuredto reflect the illumination light 16 in a first variable direction andto reflect the emission light 46 for moving the emission light spot overthe imaging plane in a second variable direction. In the depictedembodiment, the scanned illumination light 16 and the re-scannedemission light 46 travel in substantially opposite directions, i.e. theangle between the travel directions is approximately 180 degrees.Preferably, this angle is larger than 90 degrees, preferably larger than120 degrees, more preferably larger than 150 degrees so that the imagingsystem 52 and sample 32 can be positioned on either side of therotatable element. This for example allows the re-scan microscope systemto comprise only two fixed mirrors 38, 44, because no mirrors arerequired for guiding the scanned illumination light or re-scannedemission light to the sample resp, the imaging system.

FIGS. 3A, 3B and 3C illustrate that due to the rotation of the rotatableelement 25, the orientations of the first and second reflective surfaceschange causing the respective reflected light beams 16 and 46 to move aswell.

Because the reflective surfaces 24 of the rotatable element 25 do notrotate around an axis that goes through the reflective surfacesthemselves, but around a common axis 60, the respective positions of thereflective point of the illumination light 16 that is scanned and thereflection point of the emission light 46 that is re-scanned mayslightly vary. However, the applicant has found that these variations donot deteriorate the formed images outside of acceptable limits.Optionally, these variations may be compensated for by means ofappropriate post processing software.

FIGS. 4A, 4B, 4C depicts three respective time instances during a firsttime period throughout which the reflective surface 24 a scans theillumination light 16 and re-scans the emission light 46.

FIG. 4 shows an embodiment wherein the re-scan microscope system isconfigured such that, in use, during the first time period, the firstreflective surface 24 a reflects the emission light and changesorientation during said first time period for moving the emission lightspot 54 over the imaging plane 56 and is configured such that, in use,during the rotation of the rotatable element 25. In this embodiment, asecond reflective surface 24 g reflects the illumination light 16throughout a second time period and changes orientation during saidsecond time period for moving the illumination light spot 34 over and/orthrough the sample 32. In the depicted embodiment, the re-scanmicroscope system is configured such that, in use, during said secondtime period, the second reflective surface 24 g reflects emission light46 and changes orientation during said second time period for moving theemission light spot 54 over the image plane 56. Here, the second timeperiod occurs after the first time period, when the reflective surface24 g is positioned to receive the illumination light 16 and the emissionlight 46.

At respectively the first, second, third time instance (respectivelyshown in FIG. 4A. 4B, 4C), the first reflective surface 24 a is orientedsuch that the scanned illumination light 16 respectively follows path(i), path (ii), path (iii) towards the sample 32 and such thatre-scanned emission light 46 respectively follows path (iv), path (v),path (vi).

FIGS. 5A, 5B, 5C show three respective time instances during a firsttime period wherein a first reflective surface 24 a reflectsillumination light 16 and a second reflective surface 24 g reflectsemission light 46. In the depicted embodiment, reflective surfaces 24 aand 24 g are parallel, in particular oriented at 180 degrees withrespect to each other. In this embodiment, the rotatable elementcomprises a third reflective surface 24 h that is not parallel withsurface 24 a nor with surface 24 g. Reflective surface 24 h will, uponfurther rotation of the rotatable element, reflect the illuminationlight 16 for scanning, during a third time period. During the third timeperiod, another surface, in this example reflective surface 24 c, willreflect the emission light 46 for re-scanning.

FIGS. 6A and 6B respectively show the movement of illumination light 16in the primary image plane 27 and of the emission light spot 68 in theimaging plane 56.

When the illumination light 16 passes through plane 27 it is focused andhas a point spread function having a width indicated by W. The emissionlight spot 68 is focused onto imaging plane 56 and the resultingemission light spot 68 has, in the depicted embodiment, a width W/2. Asexplained above, this difference in size may be due to the lenses in thedetection optical system.

FIG. 6A shows that the illumination light 16 is scanned over the primaryimage plane 27 with a velocity in the x direction of v_(1P). As aresult, the illumination light spot 34 moves with a velocity over and/orthrough the sample in the x direction as well, yet with a lower velocityin the x direction due to the magnification caused by further lensesbetween the primary image plane 27 and the sample 32. In particular.FIG. 6A illustrates that the illumination light is scanned over theplane 27 so that the illumination light moves along several scan lines64 over plane 27. As a result, the illumination light spot is alsoscanned line by line over and/or through the sample 32. Typically, theillumination light scans over each scan line 64 in the same direction,in this example from left to right. After the illumination light hasscanned a line. e.g. line 64 a, it is moved. e.g. by an incrementalrotation of a y-axis scanner 20 a, in the −y direction, so that the nextline 64 b can be scanned. Preferably, y-axis scanner moves incrementallyin order to change the y-position of the illumination light and remainssteady when the illumination light is scanned by the rotatable elementin the x direction.

FIG. 6B shows that the emission light spot 68 is re-scanned over theimaging plane 56. Similarly, the emission light spot 68 is moved in thex direction by the rotatable element 25 over re-scan lines 66.Preferably, the illumination light 16 in primary image plane 27 and theemission light spot 68 on the imaging plane 56 move synchronously in thesense that the emission light spot 68 starts on a re-scan line 66 (onthe left hand side) at the moment the illumination light 16 starts on acorresponding scan line 64 and that the illumination light 16 andemission light spot 68 reach the end of their respective lines at thesame time.

FIG. 7A shows a polygon scanner having 12 facets. In one embodiment,some of the facets may be reflective while other facets arenon-reflective.

It should be appreciated that by increasing the number of facets, thenumber of scan lines that are written on the sample and imaging planeper rotation of the rotatable element increases. Herewith, the scanspeed may be increased.

FIG. 7B shows yet another embodiment of the rotatable element 25,wherein two mirrors 24 a, 24 g are attached to the respective ends oftwo rods 70 a, 70 g. Again, the rotatable element is configured torotate around rotational axis 60.

FIG. 8 depicts a block diagram illustrating an exemplary data processingsystem that may be used in a computing system as described withreference to FIG. 2 .

As shown in FIG. 8 , the data processing system 100 may include at leastone processor 102 coupled to memory elements 104 through a system bus106. As such, the data processing system may store program code withinmemory elements 104. Further, the processor 102 may execute the programcode accessed from the memory elements 104 via a system bus 106. In oneaspect, the data processing system may be implemented as a computer thatis suitable for storing and/or executing program code. It should beappreciated, however, that the data processing system 100 may beimplemented in the form of any system including a processor and a memorythat is capable of performing the functions described within thisspecification.

The memory elements 104 may include one or more physical memory devicessuch as, for example, local memory 108 and one or more bulk storagedevices 110. The local memory may refer to random access memory or othernon-persistent memory device(s) generally used during actual executionof the program code. A bulk storage device may be implemented as a harddrive or other persistent data storage device. The processing system 100may also include one or more cache memories (not shown) that providetemporary storage of at least some program code in order to reduce thenumber of times program code must be retrieved from the bulk storagedevice 110 during execution.

Input/output (I/O) devices depicted as an input device 112 and an outputdevice 114 optionally can be coupled to the data processing system.Examples of input devices may include, but are not limited to, akeyboard, a pointing device such as a mouse, or the like. Examples ofoutput devices may include, but are not limited to, a monitor or adisplay, speakers, or the like. Input and/or output devices may becoupled to the data processing system either directly or throughintervening I/O controllers.

In an embodiment, the input and the output devices may be implemented asa combined input/output device (illustrated in FIG. 8 with a dashed linesurrounding the input device 112 and the output device 114). An exampleof such a combined device is a touch sensitive display, also sometimesreferred to as a “touch screen display” or simply “touch screen”. Insuch an embodiment, input to the device may be provided by a movement ofa physical object, such as e.g. a stylus or a finger of a user, on ornear the touch screen display.

A network adapter 116 may also be coupled to the data processing systemto enable it to become coupled to other systems, computer systems,remote network devices, and/or remote storage devices throughintervening private or public networks. The network adapter may comprisea data receiver for receiving data that is transmitted by said systems,devices and/or networks to the data processing system 100, and a datatransmitter for transmitting data from the data processing system 100 tosaid systems, devices and/or networks. Modems, cable modems, andEthernet cards are examples of different types of network adapter thatmay be used with the data processing system 100.

As pictured in FIG. 8 , the memory elements 104 may store an application118. In various embodiments, the application 118 may be stored in thelocal memory 108, the one or more bulk storage devices 110, or apartfrom the local memory and the bulk storage devices. It should beappreciated that the data processing system 100 may further execute anoperating system (not shown in FIG. 8 ) that can facilitate execution ofthe application 118. The application 118, being implemented in the formof executable program code, can be executed by the data processingsystem 100. e.g., by the processor 102. Responsive to executing theapplication, the data processing system 100 may be configured to performone or more operations or method steps described herein.

In one aspect of the present invention, the data processing system 100may represent control module for controlling the re-scan microscopesystem as described herein.

Various embodiments of the invention may be implemented as a programproduct for use with a computer system, where the program(s) of theprogram product define functions of the embodiments (including themethods described herein). In one embodiment, the program(s) can becontained on a variety of non-transitory computer-readable storagemedia, where, as used herein, the expression “non-transitory computerreadable storage media” comprises all computer-readable media, with thesole exception being a transitory, propagating signal. In anotherembodiment, the program(s) can be contained on a variety of transitorycomputer-readable storage media. Illustrative computer-readable storagemedia include, but are not limited to: (i) non-writable storage media(e.g., read-only memory devices within a computer such as CD-ROM disksreadable by a CD-ROM drive. ROM chips or any type of solid-statenon-volatile semiconductor memory) on which information is permanentlystored; and (ii) writable storage media (e.g., flash memory, floppydisks within a diskette drive or hard-disk drive or any type ofsolid-state random-access semiconductor memory) on which alterableinformation is stored. The computer program may be run on the processor102 described herein.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising.” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of embodiments of the present invention has been presentedfor purposes of illustration, but is not intended to be exhaustive orlimited to the implementations in the form disclosed. Many modificationsand variations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the present invention.The embodiments were chosen and described in order to best explain theprinciples and some practical applications of the present invention, andto enable others of ordinary skill in the art to understand the presentinvention for various embodiments with various modifications as aresuited to the particular use contemplated.

1. A re-scan microscope system for forming an image of a sample,comprising: an illumination optical system configured to direct, andoptionally focus, illumination light at the sample therewith providingan illumination light spot at the sample, the illumination light spotcausing emission light from the sample; a detection optical systemconfigured to focus at least part of the emission light onto an imagingplane of an imaging system herewith causing an emission light spot onthe imaging plane; and a rotatable element for, when rotating, movingthe illumination light spot over and/or through the sample andsimultaneously moving the emission light spot over said imaging plane ofthe imaging system, wherein the rotatable element comprises at least twonon-parallel reflective surfaces.
 2. The re-scan microscope systemaccording to claim 1, wherein the rotatable element is configured torotate over an angle of at least 90 degrees about an axis of rotation.3. The re-scan microscope system according to claim 1, wherein therotatable element comprises a first reflective surface and a secondreflective surface, and wherein the re-scan microscope system isconfigured such that, in use, during a rotation of the rotatableelement, the first reflective surface reflects the illumination lightthroughout a first time period and changes orientation during said firsttime period for moving the illumination light spot over and/or throughthe sample.
 4. The re-scan microscope system according to claim 3,wherein the re-scan microscope system is configured such that, in use,during said first time period, the second reflective surface reflectsthe emission light and changes orientation during said first time periodfor moving the emission light spot over the imaging plane.
 5. There-scan microscope system according to claim 4, wherein the re-scanmicroscope system is configured such that, in use, during the rotationof the rotatable element, the second reflective surface reflects theillumination light throughout a second time period and changesorientation during said second time period for moving the illuminationlight spot over and/or through the sample and configured such that,during said second time period, a further reflective surface of therotatable element reflects the emission light and changes orientationduring said second time period for moving the emission light spot overthe imaging plane.
 6. The re-scan microscope system according to claim3, wherein the re-scan microscope system is configured such that, inuse, during said first time period, the first reflective surfacereflects the emission light and changes orientation during said firsttime period for moving the emission light spot over the imaging planeand such that, in use, during the rotation of the rotatable element, thesecond reflective surface reflects the illumination light throughout asecond time period and changes orientation during said second timeperiod for moving the illumination light spot over and/or through thesample.
 7. The re-scan microscope system according to claim 6, whereinthe re-scan microscope system is configured such that, in use, duringsaid second time period, the second reflective surface reflects emissionlight and changes orientation during said second time period for movingthe emission light spot over the image plane.
 8. The re-scan microscopesystem according to claim 1, wherein the rotatable element comprises arotatable polygon scanner comprising a plurality of reflective facets,wherein the first reflective surface is a first facet of the pluralityof reflective facets and the second reflective surface is a second facetof the plurality of reflective facets.
 9. The re-scan microscope systemaccording to claim 1, wherein the re-scan microscope system isconfigured to move the illumination light spot over and/or through thesample at a first velocity and move the emission light spot over theimaging plane at a second velocity, such that the second velocity isdifferent from a baseline velocity, wherein the baseline velocity isdefined as the first velocity multiplied by the optical magnification ofthe re-scan microscope system.
 10. The re-scan microscope systemaccording to claim 16, wherein the second velocity is approximatelytwice as high as the baseline velocity.
 11. The re-scan microscopesystem according to claim 9, comprising an objective configured togather the emission light from the sample and focus the emission lighton a primary image plane of the re-scan microscope system, wherein thedetection optical system is configured to image images in the primaryimage plane onto the imaging plane of the imaging system, preferablywith an optical magnification of approximately 0.5.
 12. The re-scanmicroscope system according to claim 1 that is configured such that, inuse, an angle between a travel direction of the illumination light thatis incident on the rotatable element and a travel direction of theemission light that is incident on the rotatable element for moving theemission light spot over the imaging plane is less than 90 degrees. 13.The re-scan microscope system according to claim 1, wherein therotatable element is configured to reflect the illumination light in afirst variable direction and to reflect the emission light for movingthe emission light spot over the imaging plane in a second variabledirection, wherein the angle between the first and second variabledirection is larger than 90 degrees.
 14. The re-scan microscope systemaccording to claim 1, wherein the system comprises an apertureconfigured to pass emission light and an optical system for focusing theemission light onto the aperture.
 15. A method for forming an image of asample using a re-scan microscope system, the re-scan microscope systemcomprising an illumination optical system for directing, and optionallyfocusing, illumination light at the sample therewith providing anillumination light spot at the sample, the illumination light spotcausing emission light from the sample, and the re-scan microscopesystem comprising a detection optical system focusing at least part ofthe emission light onto an imaging plane of an imaging system herewithcausing an emission light spot on the imaging plane, and a rotatableelement comprising at least two non-parallel reflective surfaces, and inthat the method comprises rotating the rotatable element for moving theillumination light spot over and/or through the sample andsimultaneously moving the emission light spot over said imaging plane ofthe imaging system.
 16. The re-scan microscope system according to claim9, wherein the second velocity is higher than the baseline velocity. 17.The re-scan microscope system according to claim 11 wherein thedetection optical system is configured to image images in the primaryimage plane onto the imaging plane of the imaging system with an opticalmagnification of approximately 0.5.
 18. The re-scan microscope systemaccording to claim 12 wherein the imaging plane is less than 60 degrees.19. The re-scan microscope system according to claim 18 wherein theimaging plane is less than 30 degrees.
 20. The re-scan microscope systemaccording to claim 13 wherein the angle between the first and the secondvariable direction is larger than 120 degrees.